Caste differentiation and division of labor are the hallmarks of social insect colonies [1, 2]. The current dogma for female caste differentiation is that female eggs are totipotent, with morphological and physiological differences between queens and workers stemming from a developmental switch during the larval stage controlled by nutritional and other environmental factors (e.g., [3-8]). In this study, we tested whether maternal effects influence caste differentiation in Pogonomyrmex harvester ants. By conducting crossfostering experiments we identified two key factors in the process of caste determination. New queens were produced only from eggs laid by queens exposed to cold. Moreover, there was a strong age effect, with development into queens occurring only in eggs laid by queens that were at least two years old. Biochemical analyses further revealed that the level of ecdysteroids was significantly lower in eggs developing into queens than workers. By contrast, we found no significant effect of colony size or worker exposure to cold, suggesting that the trigger for caste differentiation may be independent of the quantity and quality of resources provided to larvae. Altogether these data demonstrate that the developmental fate of female brood is strongly influenced by maternal effects in ants of the genus Pogonomyrmex.